Rinehart et al. have long been interested in isolating bioactive compounds from marine organisms. To this end, we have established an in-house in vitro screening program to test crude extracts for antimicrobial, antiviral, and cytotoxic activities. Once an organism with interesting biological activity has been identified, we use these assays to guide our purification of the compounds responsible for the bioactivity.
Spisula polynyma is an edible clam, which is also known as the Stimpson surf clam or the Atlantic surf clam. It belongs to the subfamily Mactrinae, family Mactridae, superfamily Mactroidea, order Veneroida, subclass Heterodonta, class Bivalvia, phylum Mollusca. S. polynyma was originally found off the coast of Japan, where it is called hokkigai and processed for sushi. It has now migrated through the Bering Strait, down past Greenland and Newfoundland, into the Atlantic ocean. The clam has a grey-white shell, 7-10 cm long. It is mainly off-white, except for the tongue which is purple in the living clam, but turns bright red after cooking.
For this invention, S. polynyma were collected, at a depth of -110 feet, from a clam bed on the eastern edge of Stellwagon bank which is located off the coast of New England, stretching from near Gloucester, Mass., north to Maine. They were shipped to us live by the New England Clam Corporation (formerly New Dawn Seafoods, Inc.) and then immediately frozen.
To test for biological activity, one clam was homogenized in 3:1 methanol/toluene. A solution of sodium chloride was added to this crude extract, causing it to separate into a toluene and an aqueous layer. The latter was further extracted with toluene, dichloromethane, ethyl acetate and 1-butanol. These extracts were all assayed against L1210 cells, where significant cytotoxicity was observed for the initial crude, toluene and dichloromethane extracts and less activity in the other three fractions. See Table I.
TABLE I ______________________________________ L1210 Cytotoxicity of Crude Extracts of S. polynyma.sup.a,b Concentration (ug/mL) Extract 250 125 50 25 12.5 5 ______________________________________ Crude 98* 98* 92 25 0 0 Toluene 100* 100* 100* 25 13 13 CH.sub.2 Cl.sub.2 100* 100* 100* 91 20 13 EtOAc 98* 98* 92* 0 0 0 1-BuOH 83 33 0 0 0 0 aqueous.sup.c 94 75 0 0 0 0 ______________________________________ Footnotes: .sup.(a) cytoxicity reported as % inhibition of growth; .sup.(b) entries marked with * showed pointed cell activity; .sup.(c) the aqueous extract was assayed at 700, 350, 140 70, 35 and 14 ug/mL.
These extracts were also assayed against Herpes simplex virus Type I (HSV-1) and CV-1 monkey kidney cells (at 100 .mu.g/6.35-mm disk), but no activity was observed. No antimicrobial activity was observed for these extract against Penicillium melinii (formerly P. atrovenetum) and Micrococcus luteus (formerly Sarcina lutea, both at 500 .mu.g/12.7-mm-disk). Later, other more purified extracts were assayed against Bacillus subtilis, Saccharomyces cerevisiae, and Escherichia coli with no bioactivity observed.